GSM8083619: 12hpf, replicate 1, scRNAseq; Oikopleura dioica; RNA-Seq
O.dioica larvae were washed several times with CASW. In the case of 8-16hpf, a 40um strainer was used for washing and collecting the animals. Meanwhile, 2ml EDTA-CASW (5Mm EDTA in calcium-free ASW) was pre-warmed at 37C for 10min before preparing the enzymatic mix. Early (4-6hpf) and late (8-16hpf) larvae were transferred to individual 1%BSA pre-soaked glass wells. After this, 2ml of filtered enzymatic cocktail (either Pronase 0,5% for early larvae, or Pronase 0,25% + Trypsin 1,25% + Cellulase 10mg/ml for late larvae, in warm EDTA-CASW) were added to each well and gently mixed at room temperature. The animals were left for 5min (for early larvae) or 10min (for late larvae). After this, 1ml of solution was removed. The remaining 1ml solution was mixed by pipetting up and down (5-10 min aprox) until all larvae were completely triturated. The enzymatic reaction was stopped by adding 3ml ice cold 0,5%BSA EDTA-CASW (enriched with 2mg/ml glycine). Dissociation was refined by pipetting up and down with a low-retention tip for 1 more minute under the microscope to avoid presence of cell aggregates. The final suspension was filtered with a 20um Pluriselect strainer into a 50ml falcon tube (previously soaked in 1%BSA-CASW). After filtering, an additional wash with 0,5%BSA EDTA-CASW was done to recover the maximum quantity of cells from the strainer. The final volume in the falcon tube did not exceed more than 20ml. The solution was centrifuged at 300G for 15 minutes, and the supernatant was discarded afterwards. The pellet was gently resuspended in 1ml 0,2%BSA MCASW (Magnesium/Calcium-free ASW). An additional wash was performed in samples from 8-16hpf due to the presence of more cellulose debris in the samples. Cell count and viability was checked using Acridine orange and Propidium iodide stain in the LUNA-FL dual fluorescence cell counter. Viability in all samples was not lower than 80% before fixation. For fixation,  a volume of 500ul of cell suspension was transferred to a 15ml falcon tube. Fixation and cryopreservation were carried out following a variation of the ACME protocol (Garcia-Castro et al., 2021) after enzymatic dissociation. Salinity of 1xPBS in ACME solution was previously increased to 32ppt using a 10X salt compensation buffer (3,42M NaCl 0,05M KCl 0,25M Na2SO4 0,32M NaHCO3) to avoid osmotic shock of dissociated cells. Cryopreserved cells were stored at -80C and posteriosly recovered and resuspended in 1%BSA 1XPBS to a concentration of 500cells/ul before proceeding with the cell barcoding. Libraries were produced following manufacturer's recommendations for the 10x Genomics Chromium controller (corresponding to the Chromium Single Cell 3' GEM Kit v3.1 user guide; CG000204 Rev D). The library quality was assessed using Qubit (ThermoFisher) and Bioanalyzer (Agilent). Eleven libraries (2x4hpf, 2x6hpf,2x8hpf, 1x11hpf, 2x12hpf and 2x16hpf) were pair-ended sequenced at the Norwegian Sequencing centre (Oslo, Norway) using Illumina Novaseq sp flowcell (2x100bp) with an expected target of 200K reads per library.